E-Letter responses to:

reports: Alok S. Shah, Yehuda Ben-Shahar, Thomas O. Moninger, Joel N. Kline, and Michael J. Welsh Motile Cilia of Human Airway Epithelia Are Chemosensory Science 2009; 325: 1131-1134 [Abstract] [Full text] [PDF]

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Published E-Letter responses:

Response to A. Pronin's E-Letter

Michael J. Welsh, Alok S. Shah, Yehuda Ben-Shahar   (28 October 2009)

Denatonium Curse

Alexey Pronin   (28 October 2009)

Response to A. Pronin's E-Letter 28 October 2009
Michael J. Welsh Department of Internal Medicine, Howard Hughes Medical Institute, University of Iowa, Iowa City, IA, Alok S. Shah, Yehuda Ben-Shahar Respond to this E-Letter: Re: Response to A. Pronin's E-Letter	The goal of our Report was to test the hypothesis that mammalian motile cilia could act as a chemosensory organelle, and we used the bitter signal transduction system as a test ("Motile cilia of human airway epithelia are chemosensory," 28 August 2009, p. 1131, published online 23 July 2009). A. Pronin indicates that correlations between T2R alleles and individuals' sensitivity to bitter tastants has been the most compelling evidence that T2R receptors detect bitter compounds and mediate the sense of taste. He goes on to say that we should do the corresponding study in airway epithelia. We agree that his work (1) and that of others (2–4) correlating T2R alleles and bitter taste sensitivity has been important. But many other discoveries indicated that T2Rs are receptors for bitter compounds [for examples see (5–10)], and a narrow focus on correlating T2R alleles and bitter taste sensitivity misses the contribution of that work, as well as important points in our Report. Although the experiment that Pronin proposes could be interesting, our current data support the conclusions of our Report. In our Report, multiple experiments revealed that T2Rs and their signal transduction machinery are located in ciliated epithelial cells and that bitter compounds elicit a cellular response. Pronin emphasizes denatonium. However, we also discovered that [Ca2+]i increased in response to salicin (100 micromolar/200 micromolar/70 micromolar), thujone (10 micromolar/-/3–30 micromolar), quinine (100 micromolar/1 micromolar/300 micromolar), and nicotine (100 micromolar/19 micromolar/100 micromolar) (parentheses indicate: concentration we tested/taste sensitivity in vivo/concentrations raising [Ca2+]i in our in vitro assays) (4, 9, 11–13). We applied denatonium at 100 micromolar, which is greater than the concentration detectable by humans. Although Pronin says that mM denatonium can cause artifacts, studies indicating that mM concentrations of denatonium stimulated hT2R4 and mT2R8 contained controls indicating that the response was not an artifact (6). Moreover, we found that denatonium only increased [Ca2+]i in epithelial cells that were ciliated (fig. S8 of our Report). More important, there need not be a close correlation between a psychophysical gustatory response in humans and a physiologic response in airway epithelia. The relationship between concentration and response could differ substantially for the perceived taste of a compound and for its stimulation of airway epithelial cells. Our findings that various T2Rs show distinct localization patterns along the cilia also raise the possibility that different T2Rs may vary in their coupling to signaling pathways. Thus, Pronin's comments about denatonium concentration might not hold for physiological or biochemical studies in lung cells. Indeed, using a biochemical assay, Pronin previously reported that 10 to 30 micromolar denatonium was required to activate T2R44 (14) and said, "Although the concentration of denatonium needed for detectable activation of hT2R44 in the GTPγS binding assay is still significantly higher than the reported 'bitter threshold' in humans, we do not expect it to match precisely human taste sensitivity. The 'bitter threshold' is a concentration at which humans begin to differentiate between water alone and water plus a compound (i.e., 'barely detectable'). Sensory detection thresholds are often much lower than observed in heterologous assays." Pronin says that we should determine whether variations in individuals' sensitivity to tasting phenylthiocarbamide (PTC) correlate with the response of ciliated airway epithelia to PTC. We have not tested PTC on airway epithelia. Although we agree that this could be an interesting study, testing for such a correlation would be technically difficult. We used primary cultures of differentiated human airway epithelial cells derived from trachea and bronchi obtained from lungs rejected for organ transplant. Obtaining cultures from individuals who were screened for PTC sensitivity would not be a trivial undertaking. In addition, interpretations can be complicated because most bitter compounds activate multiple T2Rs (4, 6, 9, 12, 15). We thank Dr. Pronin for the suggestion. Michael J. Welsh, Alok S. Shah, and Yehuda Ben-Shahar Department of Internal Medicine, Howard Hughes Medical Institute, University of Iowa, Iowa City, IA 52242, USA. References 1. A. N. Pronin et al., Curr. Biol. 17, 1403 (2007). 2. U. K. Kim et al., Science 299,1221 (2003). 3. B. Bufe et al., Curr. Biol. 15, 322 (2005). 4. B. Bufe, T. Hofmann, D. Krautwurst, J. D. Raguse, W. Meyerhof, Nat. Genet. 32, 397 (2002). 5. E. Adler et al., Cell 100, 693 (2000). 6. J. Chandrashekar et al., Cell 100, 703 (2000). 7. H. Matsunami, J. P. Montmayeur, L. B. Buck, Nature 404, 601 (2000). 8. J. Chandrashekar, M. A. Hoon, N. J. Ryba, C. S. Zuker, Nature 444, 288 (2006). 9. W. Meyerhof, Rev. Physiol. Biochem. Pharmacol. 154, 37 (2005). 10. K. L. Mueller et al., Nature 434, 225 (2005). 11. M. Behrens et al., Biochem. Biophys. Res. Commun. 319, 479 (2004). 12. A. Caicedo, E. Pereira, R. F. Margolskee, S. D. Roper, J. Neurosci. 23, 9947 (2003). 13. L. Liu, S. A. Simon, Chem. Senses 23, 125 (1998). 14. A. N. Pronin, H. Tang, J. Connor, W. Keung, Chem. Senses 29, 583 (2004). 15. M. Behrens, C. Reichling, C. Batram, A. Brockhoff, W. Meyerhof, Ann. N. Y. Acad. Sci. 1170, 111 (2009).
Denatonium Curse 28 October 2009
Alexey Pronin, Principal Scientist Senomyx, Inc., 4767 Nexus Centre Drive, San Diego, CA 92121, USA Respond to this E-Letter: Re: Denatonium Curse	In their Report ("Motile cilia of human airway epithelia are chemosensory," 28 August 2009, p. 1131, published online 23 July 2009), A. S. Shah et al. claim that the motile cilia emerging from human airway epithelial cells propel harmful inhaled material out of the lung due to activation of bitter taste receptors (T2Rs) in cilia. Because gene manipulations cannot be done in humans, the most compelling evidence that T2Rs expressed in taste buds indeed mediate bitter taste in humans came from correlating individuals' taste sensitivity with specific T2R alleles (1, 2) and/or bitter compound effects in heterologous assays (2–4). This correlation between compound taste and effects on airway epithelial cells is lacking in Shah et al.'s Report. The authors used salicin at 100 micromolar, which is below the bitter taste threshold (0.2 mM) for most people (4). In contrast, they used denatonium at 1 mM, which is 50,000 times as high as the bitter taste threshold (20 nM). But the effects on calcium concentration in airway cells were the same. The authors showed that 1 mM denatonium caused airway epithelial cell cilia to beat 10 to 25% faster, whereas at 100 micromolar it had no effect. However, even at 10 micromolar, denatonium is already so bitter to humans that people cannot distinguish between 10 and 100 micromolar solutions (5). The authors mentioned that denatonium was shown to activate hT2R4 in mM range (6). We now know that hT2R4 response is not relevant to denatonium taste, and other receptors (hT2R47) are activated by denatonium at much lower (nM to micromolar) concentrations in vitro. Physiologically active compound concentrations are largely determined by the compound's affinity to its receptor. Signaling machinery downstream from the receptor, which can vary among cell types, can have only a small effect on a cell’s sensitivity to a stimulus. If bitter taste and effects on the beat rate of the airway epithelial cell cilia were mediated by the same receptors (hT2Rs), they should be observed at similar compound concentrations, which they are not. Denatonium has been routinely used in experiments as a prototypical bitter compound. However, when used in vitro at concentrations of 1 mM or higher it causes numerous artifacts. Attributing observed effects to denatonium taste would be equivalent to attributing the effects of 1M sulfuric acid to the acid's sour taste rather than the harsh, low pH of the solution. Widespread use of this compound at such high concentrations does not make it acceptable, and more relevant compounds and/or concentrations should be used. If Shah et al. want to confirm that T2Rs mediate airway epithelial cell responses to bitter compounds, they should collect airway cells from phenylthiocarbamide (PTC)–sensitive and –insensitive individuals and demonstrate that PTC (0.1 to 0.5 mM) causes responses in cells from PTC-tasters but not from PTC-nontasters, thereby providing a correlation with bitter taste. Alexey Pronin Senomyx, Inc., 4767 Nexus Centre Drive, San Diego, CA 92121, USA. References 1. U.-K. Kim et al., Science 299, 1221 (2003). 2. A. N. Pronin et al., Curr. Biol. 17, 1403 (2007). 3. B. Bufe et al., Curr. Biol. 15, 322 (2005). 4. B. Bufe et al., Nat. Genet. 32, 397 (2002). 5. R. S. J. Keast, M. M. E. Bournazel, P. A. S. Breslin, Chem. Senses 28, 301(2003). 6. J. Chandrashekar et al., Cell 100, 703 (2000).